The overall long-term objective of the studies outlined in this proposal is an understanding of the cell and molecular mechanisms of the instability or operational reversibility of the stage of promotion in multistage hepatocarcinogenesis in the rat. Concomitant with this objective is an understanding of the mechanisms involved in the transition from the stage of promotion into later stages of hepatocarcinogenesis. Specifically this proposal is intended to characterize the natural history of those subsets of altered hepatic foci (AHF) whose continued growth is dependent on the presence of the promoting agent during the stage of promotion and the subset of AHF that are found to persist following removal of the promoting agent and thus have been termed "promoter-independent". The potential role of several biological processes in both the reversibility of some lesions and the non-regression of others will be studied in vivo. These investigations will include the potential roles of apoptosis, hepatocyte DNA synthesis, karyotypic changes, phenotypic alterations and distribution including the expression of connexins, and the transcriptional activation of proto-oncogenes and mutational changes in the p53 tumor suppressor gene in both regressing and persistent or promoter-independent AHF. Since major exogenous factors that might be involved in the development of promoter- independent AHF include both the initiating and the promoting agent, the effect of altering the dose of these agents on the quantitative yield of promoter-independent AHF, the effect of dose and known molecular effects of several different initiating and promoting agents will be investigated. The cell and molecular aspects of changes occurring in hepatocytes during initiation and promotion will be investigated in cell culture by isolation and culture of gamma-glutamyltranspeptidase-positive AHF in comparison with hepatocytes extrinsic to AHF and normal hepatocytes. The parameters to be investigated most carefully are the characteristics of the regulation of DNA synthesis and cell proliferation by promoting agents in these three cell populations as well as the development and use of a system to measure and characterize apoptosis or programmed cell death in hepatocytes of AHF. These studies should elucidate a better understanding of the mechanisms of the operational reversibility of AHF and a better understanding of mechanisms involved in the transition of hepatocytes from the stage of tumor promotion to that of tumor progression, the latter ultimately resulting in hepatocellular carcinoma.